U.S. patent application number 17/615307 was filed with the patent office on 2022-07-21 for antibacterial aminoglycoside derivatives.
The applicant listed for this patent is ZHUOHE PHARMACEUTICAL GROUP CO., LTD. Invention is credited to Shuhui CHEN, Charles Z. DING, Zhigang HUANG, Cheng LI, Dongdong TANG.
Application Number | 20220227802 17/615307 |
Document ID | / |
Family ID | 1000006290406 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220227802 |
Kind Code |
A1 |
TANG; Dongdong ; et
al. |
July 21, 2022 |
ANTIBACTERIAL AMINOGLYCOSIDE DERIVATIVES
Abstract
Disclosed are a new class of antibacterial aminoglycoside
derivatives, pharmaceutical compositions containing such compounds,
and application thereof in the preparation of drugs for treating
diseases related to bacterial infections. Specifically disclosed
are a compound represented by formula (II), pharmaceutically
acceptable salts thereof, and isomers thereof. ##STR00001##
Inventors: |
TANG; Dongdong; (Shanghai,
CN) ; HUANG; Zhigang; (Shanghai, US) ; LI;
Cheng; (Shanghai, CN) ; DING; Charles Z.;
(Shanghai, CN) ; CHEN; Shuhui; (Shanghai,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZHUOHE PHARMACEUTICAL GROUP CO., LTD |
Wuxi, Jiangsu |
|
CN |
|
|
Family ID: |
1000006290406 |
Appl. No.: |
17/615307 |
Filed: |
May 29, 2020 |
PCT Filed: |
May 29, 2020 |
PCT NO: |
PCT/CN2020/093436 |
371 Date: |
November 30, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 15/224 20130101;
A61P 31/04 20180101 |
International
Class: |
C07H 15/224 20060101
C07H015/224; A61P 31/04 20060101 A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2019 |
CN |
201910463155.1 |
Apr 16, 2020 |
CN |
202010299506.2 |
Claims
1. A compound represented by formula (II), a pharmaceutically
acceptable salt thereof, or an isomer thereof: ##STR00021##
wherein, ##STR00022## L is --O--CH.sub.2--CH.sub.2-- or
--CH.sub.2--; R.sub.1 is H or C.sub.1-3 alkyl; R.sub.2 is H,
C.sub.1-3 alkyl or ##STR00023## wherein C.sub.1-3 alkyl is
optionally substituted by 1, 2 or 3 substituent(s) independently
selected from the group consisting of F, Cl, Br, I, --OH,
--OCH.sub.3--CN, --NH.sub.2 and --NO.sub.2; R.sub.a and R.sub.b
each independently is H, --C(.dbd.O)--NH.sub.2,
--C(.dbd.O)--C.sub.1-3 alkyl or C.sub.1-3 alkyl, wherein
--C(.dbd.O)--C.sub.1-3 alkyl and C.sub.1-3 alkyl are optionally
substituted by 1, 2 or 3 R; and each R is independently F, Cl, Br,
I, --OH, --OCH.sub.3, --CN or --NH.sub.2.
2. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein the compound has a
structure represented by formula (I): ##STR00024## wherein R.sub.1
is H or C.sub.1-3alkyl; R.sub.2 is H, C.sub.1-3 alkyl or
##STR00025## wherein C.sub.1-3 alkyl is optionally substituted by
1, 2 or 3 substituent(s) independently selected from the group
consisting of F, Cl, Br, I, --OH, --OCH.sub.3, --CN, --NH.sub.2 and
--NO.sub.2; R.sub.a and R.sub.b each independently is H,
--C(.dbd.O)--NH.sub.2, --C(.dbd.O)--C.sub.1-3 alkyl or C.sub.1-3
alkyl, wherein --C(.dbd.O)--C.sub.1-3 alkyl and C.sub.1-3 alkyl are
optionally substituted by 1, 2 or 3 R; and each R is independently
F, Cl, Br, I, --OH, --OCH.sub.3, --CN or --NH.sub.2.
3. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 2, wherein the compound has a
structure represented by formula (I-1): ##STR00026## wherein
R.sub.a, R.sub.b and R.sub.1 are as defined in claim 2.
4. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein R.sub.1 is H or
--CH.sub.3.
5. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 4, wherein the compound has a
structure represented by formula (I-2): ##STR00027## wherein
R.sub.a and R.sub.b are as defined in claim 4.
6. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein each R is
independently F or Cl.
7. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein R.sub.a and
R.sub.b each independently is H, --C(.dbd.O)--NH.sub.2,
--C(.dbd.O)--CH.sub.3, --CH.sub.3 or --CH.sub.2CH.sub.3, and
wherein --C(.dbd.O)--CH.sub.3, --CH.sub.3 and --CH.sub.2CH.sub.3
are optionally substituted by 1, 2 or 3 R.
8. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 7, wherein R.sub.a and
R.sub.b each independently is H, --C(.dbd.O)--NH.sub.2,
--C(.dbd.O)--CH.sub.3, --CH.sub.3, --CH(R).sub.2,
--CH.sub.2CH.sub.3 or --CH.sub.2CH(R).sub.2.
9. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 8, wherein R.sub.a and
R.sub.b each independently is H or ##STR00028##
10. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein R.sub.2 is H,
--CH.sub.3, --CH.sub.2CH.sub.3 or ##STR00029## and wherein
--CH.sub.3 and --CH.sub.2CH.sub.3 are optionally substituted by 1,
2 or 3 substituent(s) independently selected from the group
consisting of F, Cl, Br, I, --OH, --OCH.sub.3, --CN, --NH.sub.2 and
--NO.sub.2.
11. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 10, wherein R.sub.2 is
##STR00030##
12. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 1, wherein the structure unit
##STR00031##
13. The compound, the pharmaceutically acceptable salt thereof, or
the isomer thereof according to claim 12, wherein the structure
unit ##STR00032##
14. A compound of the following formula, the pharmaceutically
acceptable salt thereof or the isomer thereof: ##STR00033##
15. A pharmaceutical composition, comprising a therapeutically
effective amount of the compound, the pharmaceutically acceptable
salt thereof, or the isomer thereof according to claim 1 as an
active ingredient, and a pharmaceutically acceptable carrier.
16. Use of the compound, the pharmaceutically acceptable salt
thereof, or the isomer thereof according to claim 1 in the
manufacture of a medicament for the treatment of bacterial
infection-related diseases.
17. The use according to claim 16, wherein the bacteria are
Carbapenem-resistant Enterobacteriaceae.
18. A pharmaceutical composition, comprising a therapeutically
effective amount of the compound, the pharmaceutically acceptable
salt thereof, or the isomer thereof according to claim 14 as an
active ingredient, and a pharmaceutically acceptable carrier.
19. Use of the compound, the pharmaceutically acceptable salt
thereof, or the isomer thereof according to claim 14 in the
manufacture of a medicament for the treatment of bacterial
infection-related diseases.
20. Use of the pharmaceutical composition according to claim 15 in
the manufacture of a medicament for the treatment of bacterial
infection-related diseases.
Description
[0001] This application claims the following priority of:
[0002] Application number CN201910463155.1 filed on May 30, 2019;
and
[0003] Application number CN202010299506.2 filed on Apr. 16,
2020.
TECHNICAL FIELD
[0004] The present invention relates to the field of medicine, in
particular to a new class of aminoglycoside derivatives,
pharmaceutically acceptable salts or isomers thereof,
pharmaceutically acceptable compositions thereof, and their use in
manufacturing a medicament for the treatment of bacterial
infection-related diseases.
BACKGROUND ARTS
[0005] A particular interest in modern drug discovery is the
development of novel small-molecular orally-bioavailable drugs that
work by binding to RNA. RNA, which serves as a messenger between
DNA and proteins, was thought to be an entirely flexible molecule
without significant structural complexity. Recent studies have
revealed a surprising intricacy in RNA structure. RNA has a
structural complexity rivaling proteins, rather than simple motifs
like DNA. Genome sequencing reveals both the sequences of the
proteins and the mRNAs that encode them. Since proteins are
synthesized using an RNA template, such proteins can be inhibited
by preventing their production in the first place by interfering
with the translation of the mRNA. Since both proteins and RNAs are
potential drug targeting sites, the number of targets revealed from
genome sequencing efforts is effectively doubled. These
observations unlock a new world of opportunities for the
pharmaceutical industry to target RNA with small molecules.
[0006] Modern biochemistry and molecular biology studies have
revealed that the binding of 30S subunit of bacterial ribosome to
tRNA is one of the key steps in protein synthesis. So far, the
crystal structures of the ribosomal 30S subunit of at least two
bacteria (Thermus thermophiles and Escherichia coli) have been
successfully reported. From the crystal structures, three sites
that bind to tRNA can be clearly identified: aminoacyl site A,
peptite site P, and E (Exit) site. Aminoglycoside medicines
specifically bind toA site of the 16S rRNA decoding region of the
30S subunit of bacterial ribosome to cause mistranslation of mRNA,
thereby interfering with protein synthesis to kill pathogenic
bacteria. Aminoglycoside medicines are highly effective
broad-spectrum antibiotics and are the most commonly used
anti-infective medicines. Most aminoglycoside medicines have
expected pharmacokinetic properties and have synergistic effects
with other anti-infective medicines, making them excellent
varieties for the treatment of life-threatening infections. In the
past few decades, many varieties of this type of antibiotics have
been clinically popular.
[0007] The history of aminoglycoside medicines originated from the
discovery of streptomycin in 1944. Later, a series of landmark
compounds (kanamycin, gentamicin, tobramycin) were successfully
launched, and the status of aminoglycoside medicines in the
treatment of gram-negative bacterial infections were established.
Between the 1970s and 1990s, the semi-synthetic aminoglycoside
antibiotics of dibekacin, amikacin, netilmicin, isepamicin and
etimicin appeared one after another, indicating that aminoglycoside
antibiotics that are effective against the bacteria resistant to
early antibiotic and have low adverse reactions can be successfully
obtained through semi-synthetic pathways, but the development of
aminoglycoside antibiotics has been slowing down. Meanwhile, people
have conducted extensive basic and clinical research on
aminoglycoside medicines, especially their bactericidal mechanism
and drug resistance mechanism, which not only gives people a deeper
understanding of this type of antibiotics, but also provides a
theoretical basis for our clinical rational use of medicines,
reducing drug-resistant bacteria, and designing new aminoglycoside
medicines against drug-resistant bacteria with these research
results.
[0008] Aminoglycoside medicines are glycosides formed by connecting
amino sugars and amino cyclic alcohols through oxygen bridges.
There are streptomycin from Streptomyces, natural aminoglycoside
medicines such as gentamicin from Micromonospora, and
semi-synthetic aminoglycoside medicines such as etimicin and
amikacin, all of which are broad-spectrum antibacterial drugs.
Aminoglycoside medicines are mainly used for systemic infections
caused by sensitive aerobic gram-negative bacteria. Although a
variety of cephalosporins and quinolones have been widely used in
clinical practice in recent years, aminoglycoside medicines are
still used for treatment of serious infections caused by aerobic
gram-negative bacteria because they have a longer PAE for common
gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella
pneumoniae, and Escherichia coli.
[0009] With the long-term and large-scale use of aminoglycoside
medicines in the clinic, serious drug resistance problems
inevitably arise in this class of medicines. At the same time, the
common side effects of aminoglycoside medicines such as ototoxicity
and nephrotoxicity also limit use of aminoglycoside medicines. In
recent years, some medicine molecules that can solve the problem of
traditional antibiotic resistance have emerged, such as the newly
developed plazomicin (WO2009067692) by Achaogen, which has
completed the third phase of clinical research.
[0010] The present invention aims to solve the problems of severe
drug resistance due to inactivating enzymes and the existence of
ototoxicity and nephrotoxicity for traditional antibiotics such as
etimicin, amikacin, gentamicin and the like. A class of novel
aminoglycoside medicines with broader antibacterial spectrum and
better activity is prepared by a simpler synthetic method compared
with the prior art.
SUMMARY OF INVENTION
[0011] The present invention provides a compound represented by
formula (II), a pharmaceutically acceptable salt thereof, or an
isomer thereof:
##STR00002##
[0012] wherein,
[0013] R' is
##STR00003##
[0014] L is --O--CH.sub.2--CH.sub.2-- or --CH.sub.2--;
[0015] R.sub.1 is H or C.sub.1-3alkyl;
[0016] R.sub.2 is H, C.sub.1-3alkyl or
##STR00004##
wherein C.sub.1-3alkyl is optionally substituted by 1, 2 or 3
substituent(s) independently selected from the group consisting of
F, Cl, Br, I, --OH, --OCH.sub.3--CN, --NH.sub.2 and --NO.sub.2;
[0017] R.sub.a and R.sub.b each independently is H,
--C(.dbd.O)--NH.sub.2, --C(.dbd.O)--C.sub.1-3alkyl or
C.sub.1-3alkyl, wherein --C(.dbd.O)--C.sub.1-3alkyl and
C.sub.1-3alkyl are optionally substituted by 1, 2 or 3 R; and
[0018] each R is independently F, Cl, Br, I, --OH, --OCH.sub.3,
--CN or --NH.sub.2.
[0019] The present invention provides a compound represented by
formula (I), a pharmaceutically acceptable salt thereof, or an
isomer thereof:
##STR00005##
[0020] wherein, R.sub.1 is H or C.sub.1-3alkyl;
[0021] R.sub.2 is H, C.sub.1-3 alkyl or
##STR00006##
[0022] wherein C.sub.1-3 alkyl is optionally substituted by 1, 2 or
3 substituent(s) independently selected from the group consisting
of F, Cl, Br, I, --OH, --OCH.sub.3, --CN, --NH.sub.2 and
--NO.sub.2;
[0023] R.sub.a and R.sub.b each independently is H,
--C(.dbd.O)--NH.sub.2, --C(.dbd.O)--C.sub.1-3 alkyl or C.sub.1-3
alkyl, wherein --C(.dbd.O)--C.sub.1-3 alkyl and C.sub.1-3 alkyl are
optionally substituted by 1, 2 or 3 R; and
[0024] each R is independently F, Cl, Br, I, --OH, --OCH.sub.3,
--CN or --NH.sub.2.
[0025] In some embodiments, the above compound has the structure
represented by formula (I-1):
##STR00007##
[0026] wherein R.sub.a, R.sub.b and R.sub.1 are as defined in the
present invention.
[0027] In some embodiments, the above R.sub.1 is H or CH.sub.3, and
the other variables are as defined in the present invention.
[0028] In some embodiments, the above R.sub.1 is H, and the other
variables are as defined in the present invention.
[0029] In some embodiments, the above compound has the structure
represented by formula (I-2):
##STR00008##
[0030] wherein R.sub.a and R.sub.b are as defined in the present
invention.
[0031] In some embodiments, each of the above R is independently F
or Cl, and the other variables are as defined in the present
invention.
[0032] In some embodiments, each of the above R is independently F,
and the other variables are as defined in the present
invention.
[0033] In some embodiments, the above R.sub.a and R.sub.b each
independently is H, --C(.dbd.O)--NH.sub.2, --C(.dbd.O)--CH.sub.3,
--CH.sub.3 or --CH.sub.2CH.sub.3, wherein --C(.dbd.O)--CH.sub.3,
--CH.sub.3 and --CH.sub.2CH.sub.3 are optionally substituted by 1,
2 or 3 R; and R and the other variables are as defined in the
present invention.
[0034] In some embodiments, the above R.sub.a and R.sub.b each
independently is H, --C(.dbd.O)--NH.sub.2, --C(.dbd.O)--CH.sub.3,
--CH.sub.3, --CH(R).sub.2, --CH.sub.2CH.sub.3 or
--CH.sub.2CH(R).sub.2, and R and the other variables are as defined
in the present invention.
[0035] In some embodiments, the above R.sub.a and R.sub.b each
independently is H or
##STR00009##
and the other variables are as defined in the present
invention.
[0036] In some embodiments, the above R.sub.2 is H, --CH.sub.3,
--CH.sub.2CH.sub.3 or
##STR00010##
wherein --CH.sub.3 and --CH.sub.2CH.sub.3 are optionally
substituted by 1, 2 or 3 substituent(s) independently selected from
the group consisting of F, Cl, Br, I, --OH, --OCH.sub.3, --CN,
--NH.sub.2 and --NO.sub.2, R.sub.a and R.sub.b and the other
variables are as defined in the present invention.
[0037] In some embodiments, the above R.sub.2 is
##STR00011##
and the other variables are as defined in the present
invention.
[0038] In some embodiments, the above structure unit
##STR00012##
and the other variables are as defined in the present
invention.
[0039] In some embodiments, the above structure unit
##STR00013##
and the other variables are as defined in the present
invention.
[0040] There are also some embodiments that come from any
combination of the above variables.
[0041] In some embodiments, the above compound is:
##STR00014##
[0042] The present invention also provides a pharmaceutical
composition, which includes a therapeutically effective amount of
the above compound, its pharmaceutically acceptable salt or its
isomer as an active ingredient, and a pharmaceutically acceptable
carrier.
[0043] The present invention also provides use of the above
compound, its pharmaceutically acceptable salt or its isomer, and
the above pharmaceutical composition in the manufacture of a
medicament for the treatment of bacterial infection-related
diseases; and In some embodiments, the above bacteria are
Carbapenem-resistant Enterobacteriaceae.
TECHNICAL EFFECTS
[0044] The present invention synthesizes the compound of formula
(II) and its isomers through a simpler preparation method, and
obtains a new class of aminoglycoside antibiotics to fight against
the drug-resistant bacterial infection caused by super bacteria
such as CRE (carbapenem-resistant Enterobacteriaceae), solving the
problems of drug resistance due to inactivation enzyme and the
existence of ototoxicity and nephrotoxicity for traditional
antibiotics. Meanwhile, the compound of the present invention has a
wider antibacterial spectrum, better activity, and no
cytotoxicity.
DEFINITIONS AND DESCRIPTIONS
[0045] Unless otherwise specified, the following terms and phrases
used herein are intended to have the following meanings. A specific
term or phrase should not be considered indefinite or unclear
without a special definition, but should be understood in its
ordinary meaning. When a trade name appears herein, it is meant to
refer to its corresponding commodity or its active ingredient.
[0046] The term "pharmaceutically acceptable" used herein refers
to, for compounds, materials, compositions and/or dosage forms,
within the scope of reliable medical judgment, suitable for use in
contact with human and animal tissues without excessive toxicity,
irritation, allergic reactions or other problems or complications,
and commensurate with a reasonable benefit/risk ratio.
[0047] The term "pharmaceutically acceptable salt" refers to a salt
of the compound of the present invention, which is prepared from a
compound discovered in the present invention with specific
substituent(s) and a relatively non-toxic acid or base. When the
compound of the present invention contains a relatively acidic
functional group, the base addition salt can be obtained by
contacting the neutral form of the compound with a sufficient
amount of base in a pure solution or a suitable inert solvent.
Pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amine or magnesium salt or
similar salts. When the compound of the present invention contains
a relatively basic functional group, the acid addition salt can be
obtained by contacting the neutral form of the compound with a
sufficient amount of acid in a pure solution or a suitable inert
solvent. Examples of pharmaceutically acceptable acid addition
salts include inorganic acid salts and organic acid salts. The
inorganic acid includes, for example, hydrochloric acid,
hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate,
phosphoric acid, monohydrogen phosphate, dihydrogen phosphate,
sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid,
etc. The organic acid includes, for example, acetic acid, propionic
acid, isobutyric acid, maleic acid, malonic acid, benzoic acid,
succinic acid, suberic acid, fumaric acid, lactic acid, mandelic
acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid,
citric acid, tartaric acid and methanesulfonic acid and the like.
Examples of pharmaceutically acceptable acid addition salts also
include salts of amino acids (such as arginine, etc.) and salts of
organic acids such as glucuronic acid. Certain specific compounds
of the present invention contain basic and acidic functional
groups, so that they can be converted into any base or acid
addition salt.
[0048] The pharmaceutically acceptable salt of the present
invention can be synthesized from the parent compound containing
acid or base radical by conventional chemical methods. Generally,
such salts are prepared by reacting these compounds in free acid or
base form with stoichiometric amounts of appropriate base or acid
in water or organic solvent or a mixture of both.
[0049] There may exist specific geometric isomers or stereoisomers
of the compounds of the present invention. The present invention
contemplates all such compounds, including tautomers, cis-isomers
and trans-isomers, (-)-enantiomers and (+)-enantiomers,
(R)-enantiomers and (S)-enantiomers, diastereomers, (D)-isomers,
(L)-isomers, their racemic mixtures and other mixtures, such as
enantiomers or diastereomeric enriched mixtures. All these mixtures
fall within the scope of the present invention. Additional
asymmetric carbon atoms may be present in substituents such as
alkyl. All these isomers and their mixtures are included in the
scope of the present invention.
[0050] Unless otherwise specified, the term "enantiomer" or
"optical isomer" refers to stereoisomers that are mirror images of
each other.
[0051] Unless otherwise specified, the term "cis-, trans-isomer" or
"geometric isomer" is caused by the inability to rotate freely due
to double bonds or single bonds of ring-forming carbon atoms.
[0052] Unless otherwise specified, the term "diastereomer" refers
to a stereoisomer in which the molecule has two or more chiral
centers and the molecules are not mirror images to each other.
[0053] Unless otherwise specified, "(D)" or "(+)" means
dextrorotation, "(L)" or "(-)" means levorotatory, and "(DL)" or
"(.+-.)" means racemic.
[0054] Unless otherwise specified, the wedge-shaped solid line bond
() and the wedge-shaped dashed line bond () are used to represent
the absolute configuration of a stereocenter, the straight solid
line bond () and the straight dashed line bond () are used to
represent the relative configuration of a stereocenter, and the
wavy line () is used to represent a wedge-shaped solid line bond ()
or a wedge-shaped dashed line bond (), or the wavy line () is used
to represent a straight solid line bond () and a straight dashed
line bond ().
[0055] The compound of the present invention may be specific.
Unless otherwise specified, the term "tautomer" or "tautomeric
form" means that at room temperature, the isomers of different
functional groups are in dynamic equilibrium and can be transformed
into each other quickly. If tautomers are possible (such as in
solution), the chemical equilibrium of tautomers can be reached.
For example, proton tautomer (also called prototropic tautomer)
includes interconversion through proton migration, such as
keto-enol tautomerization and imine-enamine tautomerization.
Valence tautomer includes mutual transformation by recombination of
some bonding electrons. A specific example of keto-enol
tautomerization is the tautomerization between two tautomers of
pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.
[0056] Unless otherwise specified, the terms "enriched in one
isomer", "rich in isomers", "rich in one enantiomer" or "rich in
enantiomers" refer to the content of one of the isomers or the
enantiomers is less than 100%, and is 60% or more, or 70% or more,
or 80% or more, or 90% or more, or 95% or more, or 96% or more, or
97% or more, or 98% or more, or 99% or more, or 99.5% or more, or
99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or
more.
[0057] Unless otherwise specified, the term "isomer excess" or
"enantiomeric excess" refers to the difference between the relative
percentages of two isomers or two enantiomers. For example, if the
content of one isomer or enantiomer is 90% and the content of the
other isomer or enantiomer is 10%, the isomer or enantiomer excess
(ee value) is 80%.
[0058] The optically active (R)- and (S)-isomers and D and L
isomers can be prepared by chiral synthesis or chiral reagents or
other conventional techniques. If an enantiomer of a compound of
the present invention is desired, it can be prepared by asymmetric
synthesis or derivatization with chiral auxiliaries, in which the
resulting diastereomeric mixture is separated, and the auxiliary
groups are removed to provide pure enantiomer desired.
Alternatively, when the molecule contains a basic functional group
(such as an amino group) or an acidic functional group (such as a
carboxyl group), it forms a diastereomeric salt with a suitable
optically active acid or base, then the diastereoisomers are
resolved by a conventional method known in the art, and the pure
enantiomers are recovered. In addition, the separation of
enantiomers and diastereomers is usually accomplished through the
use of chromatography, which employs a chiral stationary phase and
is optionally combined with chemical derivatization (for example,
the formation of carbaminate from amines). The compounds of the
present invention may contain unnatural proportions of atomic
isotopes on one or more of the atoms constituting the compound. For
example, compounds can be labeled with radioisotopes, such as
tritium (.sup.3H), iodine-125 (.sup.125I) or C-14 (.sup.14C). For
another example, deuterated drugs can be formed by substituted
hydrogen with deuterium. The bond between deuterium and carbon is
stronger than that of ordinary hydrogen and carbon. Compared with
non-deuterated drugs, deuterated drugs have advantages, such as
reducing toxic side effects, increasing drug stability, enhancing
the efficacy, and extending the biological half-life of drugs. All
changes in the isotopic composition of the compounds of the present
invention, whether radioactive or not, are included in the scope of
the present invention. "Optional" or "optionally" means that the
event or condition described thereafter may but not necessarily
occur, and the description includes the situation where the event
or condition occurs and the situation where the event or condition
does not occur.
[0059] For drugs or pharmacologically active agents, the term
"effective amount" or "therapeutically effective amount" refers to
a sufficient amount of a medicine or agent that is non-toxic but
can achieve the desired effect. For the oral dosage form of the
present invention, the "effective amount" of one active substance
in the composition refers to the amount required to achieve the
desired effect when combined with another active substance in the
composition. The determination of the effective amount varies from
person to person, depending on the age and general conditions of
the recipient, and also on the specific active substance. The
appropriate effective amount in a case can be determined by those
skilled in the art according to routine experiments.
[0060] The terms "active ingredient", "therapeutic agent", "active
substance" or "active agent" refer to a chemical entity that can
effectively treat the target disorder, disease or condition.
[0061] The term "substituted" means that any one or more hydrogen
atoms on a specific atom are replaced by substituents, and may
include deuterium and hydrogen variants, as long as the valence of
the specific atom is normal and the substituted compound is stable.
When the substituent is oxygen (i.e. .dbd.O), it means that two
hydrogen atoms are substituted. Oxygen substitution does not occur
on aromatic groups. The term "optionally substituted" means that it
can be substituted or unsubstituted. Unless otherwise specified,
the type and number of substituents can be arbitrary on the basis
that they can be chemically realized.
[0062] When any variable (such as R) occurs more than once in the
composition or structure of a compound, its definition in each
situation is independent. Thus, for example, if a group is
substituted with 0-2 R, the group can optionally be substituted
with up to two R, and R has independent options in each situation.
In addition, combinations of substituents and/or variants thereof
are permitted only when such combinations will result in stable
compounds.
[0063] When the number of a linking group is 0, such as
--(CRR).sub.0--, it indicates that the linking group is a single
bond.
[0064] When one of the variables is selected from a single bond, it
means that the two groups connected thereby are directly connected.
For example, when L in A-L-Z represents a single bond, it means
that the structure is actually A-Z.
[0065] When a substituent is vacant, it means that the substituent
is absent. For example, when X in A-X is vacant, it means that the
structure is actually A. When it is not indicated which atom the
listed substituent is connected to the substituted group, such
substituent can be bonded via any atom. For example, a pyridyl
group as a substituent can be attached to the substituted group
through any one of the carbon atoms on pyridine ring.
[0066] When the linking direction of the linking group listed is
not indicated, the linking direction is arbitrary. For example,
in
##STR00015##
the linking group L is -MW--, and -MW-- can connect ring A and ring
B in the direction same to the reading order from left to right to
form
##STR00016##
and also can connect ring A and ring B in the direction opposite to
the reading order from left to right to form
##STR00017##
The combinations of the linking groups, substituents and/or its
variants are permitted only when such combination will result in
stable compounds.
[0067] Unless otherwise specified, the term "C.sub.1-6 alkyl" is
used to represent a linear or branched saturated hydrocarbon group
containing 1 to 6 carbon atoms. C.sub.1-6alkyl includes C.sub.1-5,
C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-4, C.sub.6 and
C.sub.5alkyl, and the like. It can be monovalent (such as methyl),
divalent (such as methylene) or multivalent (such as methine).
Examples of C.sub.1-6 alkyl include, but are not limited to, methyl
(Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl
(including n-butyl, isobutyl, s-butyl and t-butyl), pentyl
(including n-pentyl, isopentyl and neopentyl), hexyl, and the
like.
[0068] Unless otherwise specified, the term "C.sub.1-3 alkyl" is
used to represent a linear or branched saturated hydrocarbon group
containing 1 to 3 carbon atoms. C.sub.1-3 alkyl includes C.sub.1-2
and C.sub.2-3 alkyl, and the like. It can be monovalent (such as
methyl), divalent (such as methylene) or multivalent (such as
methine). Examples of C.sub.1-3 alkyl include, but are not limited
to, methyl (Me), ethyl (Et), propyl (including n-propyl and
isopropyl), and the like.
[0069] The term "leaving group" refers to a functional group or
atom that can be substituted by another functional group or atom
through a substitution reaction (for example, an nucleophilic
substitution). For example, representative leaving groups include
triflate; chlorine, bromine, iodine; sulfonate groups, such as
mesylate, tosylate, p-bromobenzenesulfonate, p-tosylate, etc,
acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
[0070] The term "protecting group" includes, but is not limited to,
"amino protecting group", "hydroxy protecting group" or "sulfydryl
protecting group". The term "amino protecting group" refers to a
protecting group suitable for preventing side reactions at the
nitrogen site of the amino. Representative amino protecting groups
include but are not limited to: formyl; acyl, such as alkanoyl
(such as acetyl, trichloroacetyl or trifluoroacetyl);
alkoxycarbonyl, such as tert-butoxycarbonyl (Boc);
arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and
9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl
(Bn), trityl (Tr), 1,1-di(4'-methoxyphenyl)methyl; silyl, such as
trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and so on.
The term "hydroxy protecting group" refers to a protecting group
suitable for preventing side reactions of the hydroxyl group.
Representative hydroxy protecting groups include, but are not
limited to: alkyl, such as methyl, ethyl, and tert-butyl; acyl,
such as alkanoyl (such as acetyl); arylmethyl, such as benzyl (Bn),
p-methyloxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl
(diphenylmethyl, DPM); silyl such as trimethylsilyl (TMS) and
tert-butyldimethylsilyl (TBS) and so on. The compounds of the
present invention can be prepared by a variety of synthetic methods
well known to those skilled in the art, including the specific
embodiments listed below, the embodiments formed by their
combination with other chemical synthesis methods, and equivalent
alternatives well known to those skilled in the art. The preferred
embodiments include but are not limited to the examples of the
present invention.
[0071] The solvent used in the present invention is commercially
available.
[0072] The present invention uses the following acronyms: CFU
stands for the number of colonies; Boc stands for t-butoxycarbonyl;
MIC stands for minimum inhibitory concentration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIG. 1 shows the in vivo efficacy data of Compound 1 (at a
dose of 30 mpk) and Plazomicin (at a dose of 30 mpk) in a mouse
thigh muscle model (Enterobacteria ATCC-25922);
[0074] FIG. 2 shows the in vivo efficacy data of Compound 1 (at a
dose of 10 mpk and 30 mpk), Plazomicin (at a dose of 10 mpk and 30
mpk), and Meropenem (at a dose of 100 mpk) in a mouse pneumonia
model (Klebsiella pneumoniae ATCC-BAA-1705);
[0075] FIG. 3 shows the amplitude variations of the compound action
potential: the variations of the CAP amplitude value of Compound 1,
Gentamicin and Plazomicin at different intensities when the
frequency was fixed at 32 kHz;
[0076] FIG. 4 shows the amplitude variations of the compound action
potential: the variations of the CAP amplitude value of Compound 1,
Gentamicin and Plazomicin at different intensities when the
frequency was fixed at 16 kHz;
[0077] FIG. 5 shows the amplitude variations of the compound action
potential: the variations of the CAP amplitude value of Compound 1,
Gentamicin and Plazomicin at different intensities under a short
sound (Click);
[0078] FIG. 6 shows the damage of cochlear hair cells: A shows the
damage to inner hair cells of Compound 1, Gentamicin and
Plazomicin; and B shows the damage to outer hair cells of Compound
1, Gentamicin and Plazomicin;
[0079] FIG. 7 shows the density variations of spiral ganglion
neurons caused by Compound 1, Gentamicin and Plazomicin;
[0080] FIG. 8 shows the toxicity regression curve of Plazomicin on
HK-2 cells;
[0081] FIG. 9 shows the toxicity regression curve of Compound 1 on
HK-2 cells;
[0082] FIG. 10 shows the toxicity regression curve of Netilmicin on
HK-2 cells;
[0083] FIG. 11 shows the toxicity regression curve of Amikacin on
HK-2 cells.
DETAILED EMBODIMENTS
[0084] The present invention is described in detail through the
following examples, which are not meant any adverse limitation to
the present invention. While the present invention are described in
detail herein with its specific embodiments being disclosed,
various changes and improvements made thereto will be obvious for
those skilled in the art without departing from the spirit and
scope of the present invention.
Example 1: Compound 1
##STR00018##
[0086] Diphenylphosphinyl hydroxylamine (10 g, 42.88 mmol, 1 eq),
Compound 1-1 (13.65 g, 128.64 mmol, 12.19 mL, 3 eq) and sodium
tert-butoxide (4.95 g, 51.46 mmol, 1.2 eq) were dissolved in
tetrahydrofuran (100 mL), stirred and reacted at 5-15.degree. C.
for 16 hours. The reaction liquid was filtered, and the filtrate
was concentrated to obtain Compound 1-2.
[0087] Step 2:
[0088] Compound 1-2 (5.19 g, 42.84 mmol, 1 eq) obtained in the
previous step in tetrahydrofuran (100 mL) and
N,N-di-BOC-1H-pyrazole-1-carboxamidine (13.30 g, 42.84 mmol, 1 eq)
were stirred and reacted at 66.degree. C. for 16 hours. The
reaction liquid was cooled to room temperature, and extracted with
ethyl acetate (100 mL.times.2) after water (300 mL) was added.
[0089] The organic phases were combined, dried over sodium sulfate
and filtered. The filtrate was concentrated to obtain a crude
product, and the Compound 1-3 was obtained through column
chromatography (silica, petroleum ether/ethyl acetate=20/1, 1/1
(v/v)).
[0090] Step 3:
[0091] Compound 1-3 (1 g, 2.75 mmol, 1 eq) and 2-iodoxybenzoic acid
(847.59 mg, 3.03 mmol, 1.1 eq) were dissolved in dimethyl sulfoxide
(10 mL), and the reaction liquid was stirred at 40.degree. C. for
reaction 1 hour. The reaction liquid was filtered, and the filtrate
was extracted with tert-butyl methyl ether (20 mL.times.2 times)
after water (40 mL) was added. The organic phases were combined and
washed with saturated sodium thiosulfate (10 mL). The organic phase
was dried over anhydrous sodium sulfate, filtered and concentrated
to obtain Compound 1-4.
[0092] Step 4:
[0093] Amberlite (ion exchange resin) IRA-402(OH) (500 g) was added
to methanol (500 mL), and the solution was stirred at 20.degree. C.
for 1 hour. Then the mixture was filtered, the filter cake was
added to methanol (500 mL), and then Compound 1-5 was added into
this mixture. The mixture was stirred at 20.degree. C. for 11
hours. During the reaction, Compound 1-5 dissolved. The reaction
liquid was filtered, and the filtrate was concentrated to obtain
Compound 1-6. LCMS (ESI) m/z: 448.4 (M+1).
[0094] Step 5:
[0095] Compound 1-6 (15 g, 33.52 mmol, 1 eq) was dissolved in
methanol (150 mL), and then S-ethyl 2,2,2-trifluoroethyl thioester
(4.24 g, 26.82 mmol, 0.8 eq) in methanol (150 mL) was added
dropwise to the above methanol solution. The mixed solution was
stirred at 20.degree. C. for 16 hours. Then zinc acetate (14.72 g,
80.44 mmol, 2.4 eq) was added to the solution, and then
(N-hydroxy-5-norbornene-2,3-dicarboxyl-imido)-tert-butyl ester
(16.85 g, 60.33 mmol, 1.8 eq) and triethylamine (10.17 g, 100.55
mmol, 14.00 mL, 3 eq) in tetrahydrofuran (170 mL) were added
dropwise to the mixed solution. The reaction liquid was stirred at
20.degree. C. for 30 hours, then quenched with glycine (7 g), and
then concentrated. The concentrated liquid was diluted with
dichloromethane (1000 mL), and then washed twice with aqueous
solution of (300 mL) (water:ammonia=7:3). The organic phase was
concentrated. The crude product was purified by column
chromatography (silica, dichloromethane/methanol=50/1-5/1 (v/v),
containing a small amount of ammonia water) to obtain Compound 1-7.
LCMS (ESI) m/z: 744.3 (M+1).
[0096] Step 6:
[0097] (2S)-4-(tert-butyloxycarbonylamino)-2-hydroxy-butyric acid
(6.85 g, 31.26 mmol, 1.5 eq) was dissolved into
N,N-dimethylformamide (150 mL) and
N-hydroxy-5-norbornene-2,3-dicarboximide (5.60 g, 31.26 mmol, 1.5
eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (4.85 g,
31.26 mmol, 5.53 mL, 1.5 eq) were added to the solution. The
reaction liquid was stirred at 20.degree. C. for 2 hours, followed
by adding Compound 1-7 (15.5 g, 20.84 mmol, 1 eq) therein. The
reaction liquid was stirred at 20.degree. C. for 16 hours, then
diluted with water (200 mL), and extracted with ethyl acetate (50
mL.times.3). The combined organic phases were washed with saturated
brine (100 mL), then dried over anhydrous sodium sulfate and
filtered. The filtrate was concentrated to obtain a mixture. The
mixture was purified by column chromatography (silica,
dichloromethane/methanol=50/1-10/1 (v/v)) to obtain Compound 1-8.
LCMS (ESI) m/z: 945.5 (M+1).
[0098] Step 7:
[0099] Compound 1-8 (16.40 g, 17.35 mmol, 1 eq), di-tert-butyl
dicarbonate (4.55 g, 20.83 mmol, 4.78 mL, 1.2 eq), DIEA (2.69 g,
20.83 mmol, 3.6 mL, 1.2 eq) were dissolved in tetrahydrofuran (170
mL). Nitrogen replacement was performed for three times. The
reaction liquid was stirred at 20.degree. C. for 16 hours. The
reaction liquid was diluted with water (200 mL), and then extracted
with dichloromethane (100 mL.times.2). The combined organic phases
were washed successively with 0.1M hydrochloric acid (20 mL) and
saturated brine (60 mL), dried over anhydrous sodium sulfate, and
filtered. The filtrate was concentrated to obtain a solid mixture.
The mixture is purified by column chromatography (silica, petroleum
ether/ethyl acetate=15/1-0/1 (v/v)) to obtain the target Compound
1-9. LCMS (ESI) m/z: 1045.3 (M+1).
[0100] Step 8:
[0101] Compound 1-9 (15.00 g, 14.35 mmol, 1 eq) and ammonia water
(63.70 g, 1.82 mol, 70 mL, 126.62 eq) were dissolved in methanol
(80 mL), and the mixture was stirred at 20.degree. C. for 16 hours.
The reaction liquid was concentrated to remove the solvent, diluted
with water (100 mL), and extracted with dichloromethane (100
mL.times.3 times). The combined organic phases were washed with
saturated brine (200 mL), dried over anhydrous sodium sulfate, and
filtered. The filtrate was concentrated, and the concentrated
mixture was purified by column chromatography (silica, first
petroleum ether/ethyl acetate=10/1-0/1 (v/v), followed by
dichloromethane/methanol=6/1 (v/v), the eluent contained a small
amount of ammonia water) to obtain Compound 1-10. LCMS (ESI) m/z:
949.3 (M+1).
[0102] Step 9:
[0103] Compound 1-4 (50.63 mg, 0.15 mmol) and Compound 1-10 (145.00
mg, 0.15 mmol) were dissolved in methanol (5.00 mL), and then 4A
molecular sieve (0.5 g) was added. The mixture was stirred for 0.5
hour at 18.degree. C. under nitrogen atmosphere. Then sodium
cyanoborohydride (19.20 mg, 0.30 mmol) was added and stirred for 1
hour. The completion of the reaction was detected by LCMS. The
reaction liquid was filtered and concentrated, and separated by
preparative-IPLC: Phenomenex Synergi C18 150.times.25 mm.times.10
.mu.m; mobile phase: [water (0.225% formic acid)-acetonitrile];
acetonitrile %: 60%-90%, for 10 min to obtain Compound 1-11. LCMS
(ESI) m/z: 1294.7 (M+1).
[0104] Step 10:
[0105] Compound 1-11 (91.00 mg, 71.97 mol) was dissolved in
anhydrous dichloromethane (2.00 mL), cooled to 0.degree. C. under
nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was
added and the reaction liquid was stirred at 0-19.degree. C. for 9
hours, concentrated at room temperature, slurried with
acetonitrile/methyl tert-butyl ether (4 mL, 1/3), filtered and
concentrated to obtain Compound 1.
[0106] .sup.1H NMR (400 MHz, D.sub.2O) .delta. (ppm): 5.62 (s, 1H),
5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H),
3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m,
6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s,
3H); LCMS (ESI) m/z: 664.5 (M+1).
Example 2: Compound 2
##STR00019##
[0108] Step 1:
[0109] Compound 2-1 (1 g, 12.19 mmol, 71.94 .mu.L, 1.2 eq),
N,N-bis-BOC-1H-pyrazole-1-carboxamidine (3.15 g, 10.16 mmol, 1 eq)
and triphenylphosphine (3.20 g, 12.19 mmol, 1.2 eq) were dissolved
in tetrahydrofuran (40 mL), DIAD (2.46 g, 12.19 mmol, 2.37 mL, 1.2
eq) was added dropwise at 0.degree. C. Then the mixture was heated
to 20.degree. C. and stirred for 12 hours. Water (100 mL) was added
to the reaction solution, which was then extracted with ethyl
acetate (50 mL, 3 times). The combined organic phases were washed
with water (30 mL, 3 times), dried over anhydrous sodium sulfate,
filtered and concentrated to obtain a crude product. The crude
product was purified by chromatography column (silica, petroleum
ether/ethyl acetate=50/1 to 20/1 (v/v)) to obtain Compound 2-2.
[0110] Step 2:
[0111] Compound 2-2 (3.25 g, 8.67 mmol, 0.5 eq) was added to
Compound 1-2 (2.1 g, 17.34 mmol, 1 eq) in tetrahydrofuran (50 mL)
at 20.degree. C., and the reaction liquid was stirred at 67.degree.
C. for 12 hours. Water (100 mL) was added to the reaction liquid,
which was then extracted with ethyl acetate (50 mL.times.3). The
combined organic phases were washed with water (30 mL, 3 times),
dried over anhydrous sodium sulfate, filtered and concentrated to
obtain a crude product, which was purified by chromatography column
(silica, petroleum ether/ethyl acetate=10/1 to 1/1 (v/v)) to obtain
Compound 2-3.
[0112] Step 3:
[0113] 2-Iodoxy benzoic acid (108.09 mg, 386.02 .mu.mol, 1.1 eq)
was added to Compound 2-3 (150 mg, 350.93 .mu.mol, 1 eq) in
dimethyl sulfoxide (3 mL) at 40.degree. C. The reaction liquid was
stirred at 40.degree. C. for 2 hours. Saturated sodium
bicarbonate/sodium thiosulfate (30 mL, (v/v)) was added to the
reaction liquid, and the reaction liquid was extracted with ethyl
acetate (20 mL.times.2). The combined organic phases were washed
with saturated sodium bicarbonate/sodium thiosulfate (10 mL.times.3
times (1/1, v/v)), dried over anhydrous sodium sulfate, filtered
and concentrated to obtain Compound 2-4.
[0114] Step 4:
[0115] Compound 2-5 (118 mg, 277.37 mol, 1.1 eq) and 4A molecular
sieve (300 mg) were added to Compound 1-10 (239.32 mg, 252.16 mol,
1 eq) in 1,2-dichloroethane (2 mL) at 20.degree. C. the mixture was
stirred for 1 hour, and then sodium acetate borohydride (64.13 mg,
302.59 mol, 1.2 eq) was added. The reaction liquid was stirred at
20.degree. C. for 12 hours. with Water (20 mL) was added to the
reaction liquid, which then was extracted with dichloromethane (20
mL.times.3). The combined organic phases were washed with water (10
mL.times.3), dried over anhydrous sodium sulfate, filtered and
concentrated to obtain a crude product, which was then purified by
preparative HPLC (column: Phenomenex Synergi C18 150.times.25
mm.times.10 .mu.m; mobile phase: [water (0.225% formic
acid)-acetonitrile]; acetonitrile %: 35%-56%, 7 min) to obtain
Compound 2-6. LCMS (ESI) m/z: 1358.7 (M+1).
[0116] Step 5:
[0117] Compound 2-8 (40 mg, 29.44 .mu.mol, 1 eq) was dissolved in
dichloromethane (1 mL), and trifluoroacetic acid (1.54 g, 13.51
mmol, 1 mL, 458.70 eq) was added at 0.degree. C. The reaction
liquid was heated to 20.degree. C. and stirred for 2 hours, and
then cooled to 0.degree. C. Methyl tert-butyl ether (15 mL) was
added and the mixture was filtered, washed with methyl tert-butyl
ether (2 mL.times.3), and dried with an oil pump at 40.degree. C.
to obtain Compound 2.
[0118] .sup.1H NMR (400 MHz, D.sub.2O) .delta.=6.18-5.87 (m, 1H),
5.63 (s, 1H), 5.28-5.22 (m, 1H), 5.08 (d, J=3.8 Hz, 1H), 4.23-4.16
(m, 1H), 4.09-4.03 (m, 3H), 3.98-3.89 (m, 2H), 3.83 (br t, J=5.2
Hz, 1H), 3.79-3.68 (m, 8H), 3.46-3.38 (m, 1H), 3.33 (br d, J=13.0
Hz, 1H), 3.26-3.22 (m, 2H), 3.16-3.07 (m, 2H), 2.83 (s, 3H),
2.69-2.55 (m, 1H), 2.42-2.29 (m, 1H), 2.19-2.06 (m, 2H), 1.95-1.85
(m, 1H), 1.83-1.71 (m, 1H), 1.29-1.23 (m, 3H).
[0119] LCMS (ESI) m/z: 758.3 (M+1).
Example 3: Compound 3
##STR00020##
[0121] Step 1:
[0122] Compound 3-1 (16.00 g, 115.12 mmol) was dissolved in
acetonitrile (200.00 mL), and N--BOC-hydroxylamine (15.33 g, 115.12
mmol) and DBU (19.28 g, 126.63 mmol) were added successively. The
mixture was reacted at 11.degree. C.-25.degree. C. for 16 hours,
and then concentrated. The residue was diluted with ethyl acetate
(350 mL), washed with water (100 mL.times.3), washed with saturated
brine (100 mL) once, dried over anhydrous sodium sulfate and
concentrated. The residue was separated by column chromatography
(filler: silica gel powder, eluent: ethyl acetate/petroleum
ether=0-1/1 (v/v)) to obtain Compound 3-2.
[0123] Step 2:
[0124] Compound 3-2 (1.00 g, 5.23 mmol) and a solution of hydrogen
chloride in dioxane (10 mL, 4 mmol) were mixed together and stirred
at 20.degree. C. for 16 hours, and then concentrated under reduced
pressure to obtain Compound 3-3.
[0125] Step 3:
[0126] Compound 3-3 (581.27 mg, 6.38 mmol) and
N,N-bis-Boc-1-guanylpyrazole (1.80 g, 5.8 mmol) were dissolved in
tetrahydrofuran (20 mL) and triethylamine (1 mL) was added. The
solution was stirred at 80.degree. C. for 16 hours, and the
completion of the reaction was detected by LCMS. The mixture was
poured into water (100 mL), extracted with ethyl acetate (100
mL.times.3). The combined organic phases were washed with saturated
brine (30 mL), dried over anhydrous sodium sulfate and
concentrated. The residue was separated by column chromatography
(filler: silica gel powder, eluent: ethyl acetate/petroleum
ether=50/1-20/1 (v/v)) to obtain Compound 3-4.
[0127] Step 4:
[0128] 2-Iodoxy benzoic acid (0.34 g, 1.2 mmol) was added to
Compound 3-4 (0.40 g, 1.50 mmol) in dimethyl sulfoxide (5.00 mL),
and the mixture was stirred at 40.degree. C. for 3 hours under
nitrogen atmosphere. The reaction liquid was diluted with ethyl
acetate (100 mL), washed with water (50 mL.times.2) and saturated
brine (50 mL) and concentrated. The residue was separated by column
chromatography (filler: silica gel powder, eluent: ethyl
acetate/petroleum ether=0-1/1) to obtain Compound 3-5.
[0129] Step 5:
[0130] 4A molecular sieve (0.5 g) was added to Compound 3-5 (50.63
mg, 0.15 mmol) and Compound 1-10 (145.00 mg, 0.15 mmol) in methanol
(5.00 mL). The mixture was stirred for 0.5 hour at 18.degree. C.
under nitrogen atmosphere, and then stirred for another 1 hour
after sodium cyanoborohydride (19.20 mg, 0.30 mmol) was added. The
completion of reaction was detected by LCMS. The reaction liquid
was filtered and concentrated, and separated by preparative-HPLC:
Phenomenex Synergi C18 150.times.25.times.10 .mu.m; mobile phase:
[water (0.225% formic acid))-acetonitrile]; acetonitrile %:
60%-90%, for 10 minutes to obtain Compound 3-7.
[0131] Step 6:
[0132] Compound 3-7 (91.00 mg, 71.97 mol) was dissolved in
anhydrous dichloromethane (2.00 mL), cooled to 0.degree. C. under
nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was
added, and the reaction liquid was stirred at 0-19.degree. C. for 9
hours, concentrated at room temperature, and the residue was washed
with acetonitrile/methyl tert-butyl ether (4 mL, 1/3) to obtain
Compound 3.
[0133] .sup.1H NMR (400 MHz, D.sub.2O) .delta. (ppm): 5.62 (s, 1H),
5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H),
3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m,
6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s,
3H): LCMS (ESI) m/z: 664.5 (M+1).
Biological Activity Assay
Experimental Example 1: Detection of Antibacterial Effect of
Compound (MIC)
[0134] Three strains Enterobacteriaceae E. coli ATCC 25922, E. coli
ATCC BAA-2523, K. pneumonia ATCC BAA-1705 were used to determine
the Minimum Inhibitory Concentration (MIC) of each compound by the
micro-liquid dilution method according to the requirements of the
Institute of Clinical and Laboratory Standard (CLSI). 2-fold series
diluted compounds (with a final concentration range 0.125 g/mL-128
g/mL) were added to a round bottom 96-well plate (Catalog #3788,
Corning). A single clone of fresh bacteria on the plate of Mueller
Hinton II Agar (MHA, Cat. No. 211438, BD BBL.TM.) after overnight
culture was picked and suspended in sterile saline to adjust the
concentration to 1.times.10.sup.8 CFU/mL, and then diluted to
5.times.10.sup.5 CFU/mL by Cation-Adjusted Mueller Hinton II Broth
(MHB, Catalog #212332, BD BBL.TM.), 100 .mu.L of which was added to
the round bottom 96-well plate containing the drug. The plate was
inverted and incubated at 37.degree. C. for 20-24 hours, and the
MIC value was read. The lowest drug concentration that inhibits
bacterial growth was defined as MIC. The results are shown in Table
1.
TABLE-US-00001 TABLE 1 Antibacterial effect detection data (MIC) of
the compounds of the present invention MIC (.mu.M) K. pneumoniae E.
coli E. coli Strains ATCC BAA-1705 ATCC BAA-2523 ATCC 25922
Compound 1 4 2 2 Compound 2 4 4 2 Compound 3 0.25 1 0.5
[0135] Conclusion: The compounds of the present invention have good
in vitro antibacterial activity.
Experimental Example 2: Evaluation of Pharmacokinetics in Rats
[0136] Purpose of the Experiment:
[0137] To test the pharmacokinetic parameters of the compound of
the present invention in rats
[0138] Experimental Protocol:
[0139] 1) Experimental drug: Compound 1;
[0140] 2) Experimental animals: 3 male SD rats aged 7-9 weeks;
[0141] 3) Drug preparation: An appropriate amount of the drug was
weighed and dissolved in saline to forma 60 mg/mL solution.
[0142] Experimental Operation:
[0143] Animals were administered the drug at a dose of 150 mg/kg
and a concentration of 60 mg/mL by a single intravenous drop
infusion via the tail vein for 30 minutes. Plasma samples were
collected from the animals at 0, 0.0333, 0.0833, 0.25, 0.5, 1, 2,
4, 6, 8 and 24 hours after administration. The LC-MS/MS method was
used to determine the drug concentration in the plasma sample, and
the kinetic parameters of the tested drug are shown in Table 2:
TABLE-US-00002 TABLE 2 Pharmacokinetic evaluation results of the
compound of the present invention in rats Clearance Maximum Volume
of Area Rate Concen- Distri- Half- Under the Cl tration bution Life
Curve (mL/ C.sub.max Vd T.sub.1/2 AUC Compound Kg/min) (nM) (L/Kg)
(h) (nM h) Compound 1 9.48 315667 1.50 3.62 440373
[0144] Conclusion: The compound of the present invention has good
pharmacokinetic properties in rats.
Experimental Example 3: Study on Pharmacokinetics in Mice
[0145] Purpose of the Experiment:
[0146] The purpose of this experiment is to evaluate the
pharmacokinetic behavior of the compound after a single intravenous
injection and intragastric administration, and to investigate the
bioavailability after intragastric administration.
[0147] Experimental Operation:
[0148] CD-1 male mice aged 7 to 10 weeks were selected and treated
by intravenous administration at the dose of 1 mg/kg. The mice were
fasted for at least 12 hours before the administration, and resumed
feeding 4 hours after the administration. The mice were free to
drink during the entire experiment.
[0149] On the day of the experiment, the animals in the intravenous
group were administered with corresponding compound by a single
injection via tail vein with an administration volume of 5 mL/kg.
The animals were weighed before the administration, and the
administration volume was calculated based on the body weight. The
sample collection time was: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h.
Approximately 30 .mu.L whole blood was collected through the
saphenous vein at each time point to prepare plasma for high
performance liquid chromatography-tandem mass spectrometry
(LC-MS/MS) to determine the concentration.
[0150] All animals were subjected to euthanasia under CO.sub.2
anesthesia after the PK samples at the last time point were
collected. The non-compartmental model of the pharmacokinetic
software WinNonlin.TM. Version 6.3 (Pharsight, Mountain View,
Calif.) was used to process the data of plasma concentration, and
the linear log-trapezoidal method was used to calculate the
pharmacokinetic parameters.
[0151] Experimental results: The evaluation results of PK
properties in mice are shown in Table 3.
TABLE-US-00003 TABLE 3 Evaluation of the pharmacokinetic properties
of the compound of the present invention in mice Clearance Maximum
Volume of Area Rate Concen- Distri- Half- Under the Cl tration
bution Life Curve (mL/ C.sub.max Vd T.sub.1/2 AUC Compound Kg/min)
(nM) (L/Kg) (h) (nM h) Compound 1 15.1 7088 0.369 0.396 1924
[0152] Conclusion: The compound of the present invention has good
pharmacokinetic properties in mice.
Experimental Example 4: Experimental Evaluation of Drug Efficacy in
Mice (Mouse Thigh Muscle Model)
[0153] 12 female CD-1 mice were divided into 4 cages, 3 mice per
cage, and were injected intraperitoneally with the
immunosuppressant cyclophosphamide (150 mpk).
[0154] 24 hours later, 4 cages of mice were injected
intraperitoneally again with the immunosuppressant cyclophosphamide
(100 mpk). The strain E. coli ATCC-25922 (Enterobacteria
ATCC-25922) was recovered on a MHA plate. The recovered colonies
were picked and dissolved in saline to prepare E. coli ATCC-25922
bacterial solution with a concentration of 1.00E+07 CFU/mL for
later use in mouse thigh muscle infection. The amount of bacterial
solution injected into the thigh muscle of experimental mice was
100 L/mouse, that is, the inoculation amount was 1.00E+06
CFU/mouse. 2 h after infection, the thigh muscle tissue of the mice
in control group was taken and placed in 10 mL saline, homogenized,
and dotted on a plate with gradient dilution.
[0155] The specific administration of mice was as follows:
[0156] (1) 2 h after infection: At the end of 2 h infection, the
thigh muscle tissue of the mice in the first cage was taken and
placed in 10 mL saline, homogenized, and dotted on a plate with
gradient dilution, two duplications for each mouse. The amount of
bacteria loaded in the thigh muscle tissue of the mouse was
counted. Mice in the third and fourth cages were injected
respectively with 30 mpk Plazomicin and Compound 1
subcutaneously.
[0157] (2) 10 h after infection: Mice in the third and fourth cages
were injected respectively with 30 mpk Plazomicin and Compound 1
subcutaneously. At the end of 24 h infection, the thigh muscle
tissue of the mice in the second to fourth cages was taken and
placed in 10 mL saline, homogenized, and dotted on a plate with
gradient dilution, two duplications for each mouse. The amount of
bacteria loaded in the thigh muscle tissue of the mouse was
counted, and the experimental results were summarized and shown in
FIG. 1.
[0158] Conclusion: The results in FIG. 1 show that Compound 1 at 30
mpk has better in vivo efficacy than Plazomicin.
Experimental Example 5: Experimental Evaluation of Drug Efficacy in
Mice (Mouse Pneumonia Model)
[0159] 21 CD-1 mice were divided into 7 cages, 3 mice per cage, and
were injected intraperitoneally with the immunosuppressant
cyclophosphamide (150 mpk) on the 4th day.
[0160] On the first day, 7 cages of mice were injected
intraperitoneally again with the immunosuppressant cyclophosphamide
(100 mpk). The strain Kpn ATCC-BAA-1705 (Klebsiella pneumoniae
ATCC-BAA-1705) was recovered on a MHA plate. The recovered colonies
were picked and dissolved in saline to prepare Kpn ATCC-BAA-1705
bacterial solution with a concentration of 4.00E+08 CFU/mL for
later use in mouse lung infection. The amount of bacterial solution
infected in the lung of experimental mice was 50 .mu.L/mouse, that
is, the inoculation amount was 2.00E+07 CFU/mouse. At 2 h and 24 h
infection, the lung tissue of the mice in control group was taken
and placed in 5 mL saline, homogenized, and dotted on a plate with
gradient dilution.
[0161] The specific administration of mice was as follows:
[0162] (1) 2 h after infection: At the end of 2 h after infection,
the lung tissue of the mice in the first cage was taken and placed
in 5 mL saline, homogenized, and dotted on a plate with gradient
dilution, two duplications for each mouse. The amount of bacteria
loaded in the lung tissue of the mouse was counted. Mice in the
third and fourth cages were injected respectively with 30 mpk and
10 mpk compound Plazomicin subcutaneously, mice in the fifth and
sixth cages were injected respectively with 30 mpk and 10 mpk
compound subcutaneously, and mice in the seventh cage were injected
with 100 mpk meropenem subcutaneously.
[0163] (2) 10 h after infection: Mice in the third and fourth cages
were injected respectively with 30 mpk and 10 mpk plazomicin
subcutaneously, mice in the fifth and sixth cages were injected
respectively with 30 mpk and 10 mpk compound 1 subcutaneously, and
mice in the seventh cage were injected with 100 mpk meropenem
subcutaneously. At the end of 24 h infection, the lung tissue of
the mice in the second to seventh cages was taken and placed in 5
mL saline, homogenized, and dotted on a plate with gradient
dilution, two duplications for each mouse. The amount of bacteria
carried in the lung tissue of the mouse was counted, and the
experimental results were summarized and shown in FIG. 2.
[0164] Conclusion: FIG. 2 shows that Plazomicin and Compound 1 has
good in vivo activity in the Klebsiella pneumoniae strain 1705 lung
infection model. At the same time, the efficacy of Compound 1 is
better than that of Plazomicin, and the efficacy of Compound 1 at a
dose of 10 mpk is equivalent to that of Plazomicin at a dose of 30
mpk.
Experimental Example 6: Research Report for Auditory Safety of the
New Aminoglycoside Antibiotic Drugs
[0165] Research Purposes:
[0166] To evaluate the effects of Compound 1 and the existing
antibiotic plazomicin on auditory function in guinea pigs, and to
evaluate the auditory toxicity of Compound 1.
[0167] Research Method:
[0168] Healthy adult guinea pigs (150-250 g) were employed as the
research objects, and were randomly divided into saline control
group, gentamicin group, compound Plazomicin group and Compound 1
group, with 8 animals in each group. Subcutaneous administration is
used, and the following assays were carried out during and after
the administration for 14 consecutive days:
[0169] 1. To analyze the effects of different drugs on the auditory
function of guinea pigs, the compound action potential (CAP) of
animals was recorded on the 14th day (29th day, i.e., 4 weeks)
after administration. The results obtained were analyzed and the
changes of the threshold shift, amplitude, latency and other
indicators among different treatment groups were compared.
[0170] 2. After the different groups of animals were processed and
the auditory function data thereof were collected, the cochlea of
the animals was taken out for fixation and staining. Surface
preparation of basilar membrane of the cochlea on one side was
performed to count the loss of hair cells so as to make a cochlea
map, and the cochlea on the other side was decalcified and frozen
sectioned. The density of spiral ganglion neurons was counted and
compared among groups.
[0171] Research Results:
[0172] 1. Administration Method and Treatment
[0173] Gentamicin from Dalian Meilun Biotechnology Co., Ltd., and
Plazomicin and Compound Ifrom WuXi AppTec (WuHan) Co., Ltd. are
used, the solution of which were prepared just before use each time
by using saline for dissolution to the concentration of 50 mg/mL,
and the injection dose was 100 mg/kg body weight. Method:
subcutaneous injection, and confirmation of no liquid leakage after
each injection.
[0174] 2. Analysis of Compound Action Potential (CAP)
[0175] The compound action potential (CAP) was tested and recorded
when clicks and different frequencies of pure tones (1 KHz-32 kHz)
were applied to each group of animals, and variations in amplitude
and latency were mainly compared when clicks and medium and high
frequency pure tones (16, 32 kHz) were applied. The magnitude of
amplitude reflects the responsiveness of hair cells and auditory
nerves. The larger the amplitude and the greater the slope of the
I/O curve were, the better the responsiveness and the better the
function were. In addition, the cochlea from apical turn to basal
turn was responsive to sounds from low-frequency to high-frequency
respectively, which is the frequency correspondence of the cochlear
basilar membrane. The functional changes to different frequencies
correspond to the different structural and functional changes of
the cochlea fromapical turn to basal turn. The length of latency
was also related to the function of the hair cells and auditory
nerve response. Generally speaking, increasing of the threshold
value when the cochlea was injured would inevitably lead to the
extension of latency. In addition, the extension of latency when
the threshold value did not change significantly also reflected the
decrease in synchronicity of the auditory nerve discharge. In other
words, the extension of latency reflected the decrease of response
function. The previous ototoxicity of aminoglycoside antibiotics
was mainly concentrated in the high-frequency area. In this study,
the gentamicin group was consistent with previous results, as
summarized below.
[0176] Compound 1 only caused a decrease of CAP amplitude of the
experimental group in the high frequency (32 kHz), suggesting
hearing damage in the high frequency area, but the amplitude was
still higher than that of the Gentamicin and Plazomicin groups. The
damage of Plazomicin group occurred in a wider range, damaged at
both 16 kHz and 32 kHz, and the damage at 32 kHz greater than that
of Compound 1, but significantly lower than that of the Gentamicin
group. The damage of Gentamicin to the experimental group was
concentrated in the high frequency (32 kHz) area, the threshold
shift at 32 kHz was obvious, and the damage was more serious than
the drugs of the other two groups (see FIGS. 3 to 5).
[0177] 1) The amplitude results at 16 kHz showed that Compound 1
was no different from the Control group, while the Plazomicin group
had 25.9% damage.
[0178] 2) The CAP amplitude results at 32 kHz showed that Compound
1, Plazomicin and Gentamicin caused 34.7%, 48.2% and 74.3% hearing
damage at 32 kHz, respectively, that is, Compound 1 still caused
hearing damage at 32 kHz, which however was reduced by 13.5% and
39.6% respectively compared to Plazomicin and Gentamicin.
[0179] 3) The CAP amplitude under click indicated that both
Compound 1 and Gentamicin were consistent with the Control group,
while Plazomicin caused hearing damage.
[0180] The specific values were as follows:
[0181] 1) CAP amplitude at 16 kHz: Two-way ANOVA (Holm-Sidak
method) showed that there were differences between the four groups
of animals, F3, 570=7.858, p<0.001. Among them, there was no
statistical difference in hearing between animals in the Compound 1
group, the Control group, and the Gentamicin group. The hearing of
animals in the Plazomicin group was lower than that in the Control
group (t=4.566, p<0.001), Gentamicin group (t=4.099, p<0.001)
and Compound 1 group (t=2.799, p=0.021) respectively. *: p<0.05.
The response of each group was maximum at 90 dB, at which the
hearing of animals in Compound 1 group (381.646.+-.20.895 uv) was
significantly higher than that in the Plazomicin group
(282.058.+-.22.569 uv, t=2.799, p=0.021) and not significantly
different from the Control group (383.130.+-.19.545) and Gentamicin
group (373.329.+-.15.332 uv). 2) CAP amplitude at 32 kHz: Two-way
ANOVA (Holm-Sidak method) showed that there are differences between
the four groups of animals, F3, 570=100.611, p<0.001. The
hearing of animals in the Compound 1 group was lower than that in
the Control group (t=5.019, p<0.001), higher than the Plazomicin
group (t=3.128, p=0.002) and Gentamicin group (t=10.484,
p<0.001). The response of each group was maximum at 90 dB, at
which, the hearing of the animals in the Compound 1 group
(79.420.+-.7.000 uv) was lower than that in the Control group
(121.608.+-.6.548 uv, t=4.401, p<0.001), higher than that in the
Gentamicin group (31.272.+-.5.137 uv, t=5.545, p<0.001) and
Plazomicin group (62.982.+-.7.561 uv, t=1.595, p=0.111).
[0182] 3) CAP amplitude under click: Two-way ANOVA (Holm-Sidak
method) showed differences among the four groups of animals, F3,
570=6.751, p<0.001. The hearing of animals in the Compound 1
group was significantly better than that in the Plazomicin group
(t=3.493, p=0.003), and not significantly different between the
Control group and the Gentamicin group.
[0183] In general, the results of CAP amplitude proved that the
hearing damage of Compound 1 to experimental animals was
significantly lower than that of Gentamicin and Plazomicin.
[0184] 3. Variations in the Number of Hair Cells
[0185] In order to compare the effects of different drugs on hair
cells, hair cell staining and counting on the whole basilar
membrane of the cochlea are performed. The results indicated that
the Gentamicin group had 12-67.7% loss of outer hair cells in the
medium and high frequency region (60-100% from the apical turn) and
the loss was more obvious in the high frequency area. The
Plazomicin group had 11.2-28.1% loss of outer hair cells in the
high frequency (70-100% from the apical turn) and 16.7-24.2% loss
of the outer hair cells at the beginning of the apical turn
(10-20%). However, in the Compound 1 group, the loss of outer hair
cells only occurred in the low-frequency region (from the top turn
-40%), in which the loss rate of outer hair cells was about
2.5-11%, and the outer hair cells were relatively intact in the
high frequency area (see FIG. 6 B). The specific values are shown
in Table 4.
[0186] In the Compound 1 group, the inner hair cells were almost
undamaged. Both the Plazomicin and Gentamicin groups had
3.5.+-.3.0% and 9.3.+-.4.1% loss of inner hair cells near the end
of the basal turn (100% from the apical turn) respectively (see
FIG. 6 A). The specific values are shown in Table 5.
[0187] In summary, the Compound group only had a slight loss of
outer hair cells in the apical turn, and the rest part especially
the basal turn and inner hair cells were preserved intact. The hair
cell toxicity of Compound 1 was significantly lower than that of
the Gentamicin and the Plazomicin.
[0188] 4. Variations of Spiral Ganglion Neurons
[0189] The cochlea of the guinea pig was defined as Turn 1, Turn 2,
Turn 3, and Turn 4 from the basal to the apical turn. The spiral
ganglion neurons (SGNs) were stained with TuJ on frozen sections,
and the density thereof in a specific area were counted and
compared between groups. There was no significant difference for
the SGN density in each turn between the Compound 1 group and the
Control group, that is, there was no damage to spiral ganglion
neurons. The Gentamicin group had obvious damage in each turn. The
Plazomicin group had a decrease in SGN density near the apical
turn, but it was better than Gentamicin group. Through the two-way
ANOVA, there was a significant difference between the groups, F(3,
74)=35.43, p<0.0001 (see FIG. 7). The specific values are shown
in Table 6.
[0190] Conclusion: Based on the analysis of compound action
potentials between different groups, it was confirmed that the
Compound 1 group had hearing damage only at high frequency (32
kHz), which was better than the Plazomicin and Gentamicin groups.
The observation of hair cells and spiral ganglion neurons confirmed
that except for 2.5-11% loss of the outer hair cells near the
apical turn, Compound 1 did not cause obvious damage to the outer
hair cells in other areas, and the number of inner hair cells and
the number of spiral ganglion neurons were not affected, which was
significantly better than Gentamicin and Plazomicin groups.
Therefore, Compound 1 was administered subcutaneously in animals
(guinea pigs) for 14 consecutive days, and the ototoxicity thereof
was less than that of the Plazomicin and Gentamicin after another
14 days. Based on the results of this study, it was confirmed that
Compound 1 obtained by the present invention was better than
Plazomicin and Gentamicin in terms of the auditory toxicity.
TABLE-US-00004 TABLE 4 Damage of outer ear hair cells on Day 29 (%,
percentage) Damage percentage (%) Compound 1 Plazomicin Gentamicin
10 11.0 .+-. 1.3 24.4 .+-. 13.5 9.5 .+-. 2.9 20 6.3 .+-. 0.9 16.7
.+-. 13.4 4.1 .+-. 0.7 30 6.2 .+-. 1.1 10.5 .+-. 6.5 2.7 .+-. 0.7
40 2.5 .+-. 1.4 7.8 .+-. 4.0 3.2 .+-. 1.0 50 1.1 .+-. 0.8 7.2 .+-.
2.0 3.2 .+-. 1.5 60 1.4 .+-. 1.5 5.7 .+-. 5.1 12.6 .+-. 5.6 70 0.7
.+-. 0.3 11.2 .+-. 11.8 13.6 .+-. 6.1 80 0.5 .+-. 0.3 19.9 .+-.
15.7 12.0 .+-. 5.4 90 0.2 .+-. 0.3 20.7 .+-. 7.9 42.8 .+-. 8.2 100
0.9 .+-. 0.7 28.1 .+-. 10.1 67.7 .+-. 9.8
TABLE-US-00005 TABLE 5 Damage of inner hair cells on Day 29 (%,
percentage) Damage percentage (%) Compound 1 Plazomicin Gentamicin
10 0.3 .+-. 0.3 0.5 .+-. 0.4 0 20 0 0.3 .+-. 0.3 0 30 0 0.5 .+-.
0.5 0 40 0.1 .+-. 0.1 0.2 .+-. 0.2 0 50 0 0 0 60 0 0.8 .+-. 0.8 0.4
.+-. 0.4 70 0.3 .+-. 0.3 1.3 .+-. 1.3 0.6 .+-. 0.6 80 0 0 0.2 .+-.
0.2 90 0 0 2.5 .+-. 1.7 100 0.7 .+-. 0.5 3.5 .+-. 3.0 9.3 .+-.
4.1
TABLE-US-00006 TABLE 6 Density variation of spiral ganglion neurons
on Day 29 (n/10000 .mu.m.sup.2) Turn Gentamicin Plazomicin Compound
1 Control Turn 4.407 .+-. 0.517 6.816 .+-. 0.852 8.230 .+-. 0.500
7.548 .+-. 0.534 1 Turn 5.540 .+-. 0.757 5.876 .+-. 0.536 8.282
.+-. 0.254 7.695 .+-. 0.298 2 Turn 4.604 .+-. 0.598 5.553 .+-.
0.793 8.136 .+-. 0.247 7.935 .+-. 0.292 3 Turn 5.259 .+-. 0.280
5.641 .+-. 0.767 7.469 .+-. 0.772 6.936 .+-. 0.205 4
Experimental Example 7: Toxicity Test of the Compounds of the
Present Invention on HK-2 Cells
[0191] Cell Preparation:
[0192] On the day of the experiment, when the HK-2 cells in the
culture flask reached 80%-90% confluent, the culture medium was
discarded, the cells were washed twice with Dulbecco's phosphate
buffered saline (DPBS) and digested for 1 to 2 minutes with 3 mL
trypsin (T150 cell culture flask), and immediately 9 ml complete
medium (RPMI1640+10% FBS) were added to terminate the digestion.
After termination, single cells suspension were formed by pipetting
gently, which were centrifuged at 1000 revolutions per second for 5
minutes. The supernatant was discarded, and fresh complete medium
were added, and the cells were pipetted evenly. The actual cell
density was measured according to the cell counter and the cell
suspension was adjusted to 2.5.times.10.sup.5 cells/mL. 80 .mu.L of
cell suspension was drawn by a row pipettor and added into a
96-well black bottom plate (2.times.10.sup.4 cell/well), and then
incubated in a carbon dioxide incubator for 4.5 hours, which was
defined as a cell plate.
[0193] Preparation of Compounds:
[0194] a. The compound mother liquor was prepared according to the
following table, with complete medium as the solvent;
TABLE-US-00007 TABLE 7 Compound Information Mass Purity
Concentration Volume Compound (mg) (%) (mg/mL) (.mu.L) Plazomicin
5.88 99.65 50 117.19 Compound 1 5.51 95 50 104.69 Netilmicin 5.00
-- 50 100.00 Amikacin 5.74 -- 50 114.80
[0195] b. 50 microliters of complete medium was added into columns
3-11 in the 96-v well plate;
[0196] c. 75 microliters of test compound (50 mg/mL) and positive
control were added to the second column of the 96-v well plate;
[0197] d. 25 microliters of compound was drawn from the second
column and added into the third column, blown and sucked a few
times by a row pipettor, then 25 microliters of liquid was drawn
from the third column and added to the fourth column, and
subsequently subjected to a 3-fold series dilution until the 10th
column. From column 2 to column 11, the compound concentration was
50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.008, 0 mg/mL;
[0198] e. 20 .mu.L compound solutions of various concentrations
prepared were transferred by a row pipettor to the corresponding
wells of the cell plate, which was defined as a testing plate.
[0199] Culture of the Testing Plate:
[0200] All the plates were incubated in an incubator at 37.degree.
C., 5% CO.sub.2 for 43 hours.
[0201] Reading:
[0202] After incubation, 10 microliters of Alma Blue was added to
the testing plate. The testing plate was immediately incubated in
an incubator at 37.degree. C., 5% CO.sub.2 for 3 hours. Then the
fluorescence value of each well of the testing plate was read by a
microplate reader (wavelength Ex 540 nm/Em 585 nm). Then prism
software was used to simulate the curve to calculate CC.sub.50
value.
[0203] Research Results:
TABLE-US-00008 TABLE 8 Experimental results and predicted CC.sub.50
CC.sub.50 of compound on CC.sub.50 of Maximum cells predicted by
HK-2 cell inhibition software (Prism) Compound (mg/mL) rate (%)
(mg/mL) Plazomicin >10 35.98 19.53 Compound 1 >10 21.96 113.9
Netilmicin 10.64 48.73 10.64 Amikacin 8.157 68.40 8.156
[0204] Conclusion: The toxicity of compound 1 and Plazomicin to
HK-2 cells was significantly lower than that of Netilmicin and
Amikacin. In combination with the toxicity regression curve and
software prediction (FIG. 8-11), the toxicity of Compound 1 to HK-2
cells was lower than Plazomicin.
* * * * *